Studies of the genome are now entering a phase focusing on comparative and functional genomics. Of significant importance is the resolving of the evolutionary history of the human genome as it relates to both genome organization and chromosomal architecture. Comparative genome analysis offers the potential to interpret the evolutionary dynamics of gene organization within chromosomes, to reveal the forces governing conservation of synteny, and to specify the adaptive rationale behind genome organization. Chromosomal instability is a hallmark of cancer cells. Similarly, during the course of mammalian chromosome evolution a tremendous degree of reshuffling has occurred. The next challenge for comparative genome analysis is to better understand the patterns and rates of chromosomal evolution, and apply these to reveal the processes involved in breaking and reshaping genomes. The technology and resources of the Comparative Molecular Cytogenetics Core, technology unique to the NCI and the entire NIH, combined with resources of the Genetics Branch, the Cancer Chromosome Aberration Project (CCAP) and the Laboratory of Genomic Diversity present significant new opportunities to map chromosomal breakpoints involved with cancer, tumorigenesis and evolution. The ability to flow sort chromosomes present in the Core combined with microarray technology would expedite the identification and cloning of genes at the site of genomic rearrangements in both disease and evolution. A number of hypotheses can be tested by the research. Among the more important are: (1) that genome rearrangements do not represent random events but instead higher order genome architectural features;(2)that chromosomal rearrangements are primary lesions or responsible for progression in tumorigenesis can be tested; and (3) that chromosomal rearrangements seen in evolution and disease are related. A future application of the research results would foresee the use of genome changes seen in both disease and evolution as biomarkers of cancer risk, diagnostics or prognostic assessments. Rearranged chromosomes fixed in evolution or aberrant chromosomes from cancer cell lines can be sorted in high numbers. The DNA can then be extracted and hybridized to DNA microarrays to determine breakpoints with a degree of accuracy and ease previously unknown. High numbers of chromosomes can also be sorted and the DNA purified to establish DNA libraries. The CCAP already provides a collection of high-resolution fluorescence in situ hybridization(FISH)-mapped bacterial artificial chromosome (BAC) clones that are anchored on the sequence of the human genome linking the cytogenetic and physical maps of the human genome. An analogous set of murine clones is anticipated. Our collaborators at Cold Springs Laboratory now print human BAC clone arrays. These BAC arrays provide a hybridization platform that will allow assessment of regional genome copy number changes (gains and losses) at a 1-2 Mb resolution. The use of such arrays would allow a high definition of genome changes in human cancers, precancers, and analogously in murine cancer models. Intra-chromosomal rearrangements can be identified using FISH of DNA probes of decreasing size. Chromosome painting probes can be made by chromosome sorting and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) while painting probes specific to single chromosomal bands may be generated by chromosomal laser microdissection. Each band of the human karyotype can now be defined by cloned DNA. Thus, comparative molecular cytogenetics should be able to characterize any breakpoint in both evolution and disease. Recent large-scale comparative sequencing efforts have begun to provide some insight into chromosome breakpoints. There is now ample evidence that genomic features flanking the sites of recombination may result in susceptibility to chromosome rearrangements. For instance, the most frequent constitutional translocation in humans the t(11;22)(q23;q11) is due to a highly specific Alu-mediated recombination. Other sequences such as transposons, minisatellites and duplicons may be responsible for promoting chromosome rearrangements. Fragile sites, which often appear to be associated with break points in chromosomal rearrangements in both cancer and evolution, may contain such repeat sequences. Genome rearrangements do not represent random events but instead higher order genome architectural features. An increasing number of diseases is now associated with unstable genomic regions. The clinical phenotype is a consequence of abnormal dosage of genes located within the rearranged fragments. These disease are know as genomic disorders and they have wide-ranging effects on human health as detailed in recent reviews. Chromosome-specific low-copy repeats or duplicons occur in multiple regions of the human genome and that these sequences facilitate chromosome rearrangements. Sequence homology between duplicons provides a chance for misalignment during meiosis, leading to unequal exchange and chromosome rearrangements such as deletions, duplications, inversions, and inverted duplications, depending on the orientation of the recombining duplicons. This process and the subsequent divergence of duplicated segments are essential to the generation of diversity and new genes over evolutionary time; although the more typical, short-term effect is genetic disease. Detailed analysis of the structure, polymorphic variation and mechanisms of recombination in genomic disorders as well as the evolutionary origin of various duplicons will further our understanding of the structure, function and fluidity of the human genome. The presence of transposable elements is hypothesized to have profound effect on genome structure and rearrangements leading to disease or evolution. Mobile elements have been shown to be involved in about half the chromosome rearrangements in Drosophila. It has long been suggested that a correlation between fragile sites (FSs) and oncogenes (OGs) may be involved in the genesis and progression of tumors. A relationship has also been proposed between the location of human FSs, cancer breakpoints, OGs and the evolutionary rearrangements of chromosomes. Recently, the common fragile site FRA16D which contains a mutated minisatellite has been shown to be involved with multiple myeloma translocations and head and neck cancer. However, the evolutionary role of FSs and OGs is not yet well understood. Clearly, the available data are completely insufficient to seriously test whether fragile sites (FSs) and oncogenes (OGs) are commonly involved in tumorigenesis and whether they could eventually be used as biomarkers for cancer prognosis. Centrosome size and centrosome number were recently shown to be positively correlated with aneuploidy and chromosome instability. Studies of invasive breast tumors show that up to 80% are aneuploid and that even a larger percentage exhibit amplified centrosomes. These results suggest that centrosome amplification may drive chromosome instability and have diagnostic value as well as a potential target for cancer therapy. In colorectal cancer loss of chromosome 18q21 is well documented. It is clear that cytogenetics combined with molecular techniques is a powerful tool to investigate mechanism of genome instability in cancer and evolution. The results of such studies may allow us to determine the mechanisms and lesions that lead to certain cancers. This knowledge determines what are good biomarkers for cancer diagnosis at the molecular cytogenetic level and may lead to improved diagnostics and treatment.
